Study On The Expression And Role Of FTO During Early Follicular Development In Mice

BackgroundThe follicle is the basic reproductive unit of the ovary,which consists of an oocyte and surrounding granulosa cells.A dormant follicular pool consisting of a large number of resting primordial follicles has been established at birth in female mammals.After puberty,follicles begin to develop,which is an irreversible,orderly and periodic complex biological process.The development outcome includes atresia or dominant follicles,which directly determines the ovarian function and fertility of women.Once the abnormal activation and development disorder of follicles,it will cause dysfunction of female reproductive system,and eventually lead to fertility problems.Polycystic ovary syndrome and premature ovarian insufficiency are common reproductive endocrine diseases associated with follicular maldevelopment.Fat mass and obesity-associated protein(FTO),also known as ALKBH9.With demethylase activity,the substrate was represented by m6A modification with the highest abundance in mRNA.By regulating m6A modification,FTO extensively regulates cellular biological processes.Studies have shown that FTO and m6A modifications regulate the progress of reproductive process,and are closely related to reproductive endocrine diseases related to ovarian reserve and follicular development disorder in women.However,the regulatory role of FTO on follicular development is not yet clear.Therefore,in this study,the ovaries of newborn mice were used as research materials to clarify the expression pattern of FTO in the early follicular development of mice and to explore the role and potential mechanism of FTO in early follicular development.Objective:1.To determine the expression rule of FTO during early follicular development in mice;2.To clarify the expression and cell localization of FTO in follicles at different developmental stages;3.To explore the effect of FTO on early follicular development and its potential mechanism.Materials and methods:In this study,the expression levels of FTO mRNA and FTO protein in ovary of newborn mice of different birth days were detected by real-time fluorescence quantitative PCR and Western blotting.Immunohistochemical staining was used to analyze the expression and cell localization of FTO in follicles at different developmental stages.The expression level of FTO in ovary of mice was upregulated by lentivirus infection,and ovarian tissue was cultured in vitro for HE staining to observe follicular development.The expression levels of follicular development-related genes in ovary were detected by real-time fluorescence quantitative PCR.The apoptosis of ovarian cells was analyzed by TUNEL staining.Results:1.The expression level of FTO was low in the ovaries of mice at the postnatal day 1,high in the ovaries at the postnatal day 5 and 10,and then decreased to a low level.With the increase of the age of mice,the trend first increased and then decreased;2.FTO was continuously expressed at each stage of follicular development in mice,and was mainly located in follicular granulosa cells;3.After overexpression of FTO,the development of primary ovarian follicles in the ovaries of mice at the postnatal day 5 was relatively stagnant,while the primordia follicles in the ovaries of mice at the postnatal day 1 were mostly in a resting state,indicating that the overexpression of FTO slowed down the development of early follicles.4.FTO overexpression up-regulated the expression levels of follicular development-related genes AMH mRNA and AMHR Ⅱ mRNA(P<0.05);5.Different degrees of ovarian cell apoptosis were observed in the process of in vitro culture,and the overexpression of FTO inhibited the cell apoptosis of early follicles.Conclusion:1.FTO is expressed in granulosa cells of mouse follicles at all developmental stages,and increased firstly and then decreased during early follicular development;2.Overexpression of FTO delays early follicular development and may affect follicular development by regulating the expression of AMH and AMHR II.3.Overexpression of FTO inhibits apoptosis of early follicular cells,which may play a potential role in maintaining early follicular survival.